Differential RNA accumulation of two β-tubulin genes in arbuscular mycorrhizal fungi

RNA was isolated from spores of different arbuscular mycorrhizal (AM) fungi and used for RT-PCR with degenerate primers for beta-tubulin genes. PCR products were cloned and the sequence of several clones was analysed for each fragment. Comparison of sequences identified two loci for beta-tubulin genes with different GC content and codon usage. Btub1 sequences were most similar to beta-tubulin genes from the Oomycota, while Btub2 sequences showed highest similarity to sequences from the Zygomycota. RT-PCR experiments were carried out to monitor RNA accumulation patterns of Btub1 and Btub2 in asymbiotic germinating spores and in symbiotic extraradical hyphae of three different AM fungi. This indicated that Btub1 is constitutively expressed in Gigaspora rosea, but down-regulated during symbiosis in Glomus mosseae and Glomus intraradices. In contrast, Btub2 showed constitutive expression in the two Glomus species, but down-regulation in G. rosea. Further analysis of different fungi indicated that Btub2 primers could be used to specifically monitor RNA accumulation of AM fungi in environmental samples.     

Specific β-tubulin isotypes can functionally enhance or diminish epothilone B sensitivity in non-small cell lung cancer cells

Epothilones are a new class of microtubule stabilizing agents with promising preclinical and clinical activity. Their cellular target is β-tubulin and factors influencing intrinsic sensitivity to epothilones are not well understood. In this study, the functional significance of specific β-tubulin isotypes in intrinsic sensitivity to epothilone B was investigated using siRNA gene knockdown against βII-, βIII- or βIVb-tubulins in two independent non-small cell lung cancer (NSCLC) cell lines, NCI-H460 and Calu-6. Drug-treated clonogenic assays showed that sensitivity to epothilone B was not altered following knockdown of βII-tubulin in both NSCLC cell lines. In contrast, knockdown of βIII-tubulin significantly increased sensitivity to epothilone B. Interestingly, βIVb-tubulin knockdowns were significantly less sensitive to epothilone B, compared to mock- and control siRNA cells. Cell cycle analysis of βIII-tubulin knockdown cells showed a higher percentage of cell death with epothilone B concentrations as low as 0.5 nM. In contrast, βIVb-tubulin knockdown cells displayed a decrease in epothilone B-induced G(2)-M cell cycle accumulation compared to control siRNA cells. Importantly, βIII-tubulin knockdowns displayed a significant dose-dependent increase in the percentage of apoptotic cells upon treatment with epothilone B, as detected using caspase 3/7 activity and Annexin-V staining. Higher concentrations of epothilone B were required to induce apoptosis in the βIVb-tubulin knockdowns compared to control siRNA, highlighting a potential mechanism underlying decreased sensitivity to this agent. This study demonstrates that specific β-tubulin isotypes can influence sensitivity to epothilone B and may influence differential sensitivity to this promising new agent.     

Catabolite repression of β-galactosidase synthesis in Escherichia coli

1. Repression by glucose of β-galactosidase synthesis is spontaneously reversible in all strains of Escherichia coli examined long before the glucose has all been consumed. The extent of recovery and the time necessary for reversal differ among various strains. Other inducible enzymes show similar effects. 2. This transient effect of glucose repression is observed in constitutive (i⁻) and permease-less (y⁻) cells as well as in the corresponding i⁺ and y⁺ strains. 3. Repression is exerted by several rapidly metabolizable substrates (galactose, ribose and ribonucleosides) but not by non-metabolized or poorly metabolized compounds (2-deoxyglucose, 2-deoxyribose, phenyl thio-β-galactoside and 2-deoxyribonucleosides). 4. The transient repression with glucose is observed in inducible cells supplied with a powerful inducer of β-galactosidase synthesis (e.g. isopropyl thio-β-galactoside) but not with a weak inducer (lactose); in the latter instance glucose repression is permanent. Diauxic growth on glucose plus lactose can be abolished by including isopropyl thio-β-galactoside in the medium. 5. In some strains phosphate starvation increases catabolite repression; in others it relieves it. Adenine starvation in an adenine-requiring mutant also relieves catabolite repression by glycerol but not that by glucose. Restoration of phosphate or adenine to cells starved of these nutrients causes a pronounced temporary repression. Alkaline-phosphatase synthesis is not affected by the availability of adenine. 6. During periods of transient repression of induced enzyme synthesis the differential rate of RNA synthesis, measured by labelled uracil incorporation in 2min. pulses, shows a temporary rise. 7. The differential rate of uracil incorporation into RNA falls during exponential growth of batch cultures of E. coli. This is equally true for uracil-requiring and non-requiring strains. The fall in the rate of incorporation has been shown to be due to a real fall in the rate of RNA synthesis. The significance of the changes in the rate of RNA synthesis is discussed. 8. A partial model of catabolite repression is presented with suggestions for determining the chemical identification of the catabolite co-repressor itself.     

Expression of class III β-tubulin in normal and neoplastic human tissues

 The class III β-tubulin isotype is widely used as a neuronal marker in normal and neoplastic tissues. This isotype was, however, also immunodetected in certain tumours of non-neuronal origin such as squamous cell carcinoma. Using a newly described monoclonal antibody we compared the distribution of class III β-tubulin in normal and neoplastic tissues. The TU-20 mouse monoclonal antibody was prepared against a conserved synthetic peptide from the C-terminus of the human class III β-tubulin isotype, and its specificity was confirmed by immunoblotting, by competitive enzyme-linked immunosorbent assay and by immunofluorescence microscopy on cultured cells. In different cell lines of various origins the antibody reacted only with neuroblastoma Neuro-2a cells and with embryonal carcinoma P19 cells stimulated to neuronal differentiation by retinoic acid. Immunohistochemistry on formaldehyde-fixed paraffin-embedded normal human tissues revealed the presence of the class III β-tubulin isotype in cell bodies and processes of neuronal cells in the peripheral and central nervous systems. In other tissues, this β-tubulin isotype was not immunodetected. Class III β-tubulin was found in all cases of ganglioneuroblastoma, ganglioneuroma, medulloblastoma, neuroblastoma, sympathoblastoma and in one case of teratoma. In contrast, no reactivity was detected in tumours of non-neuronal origin, including 32 cases of squamous cell carcinoma. The results indicate a specific TU-20 epitope expression exclusively in neuronal tissues. The antibody could thus be a useful tool for the probing of class III β-tubulin functions in neurons as well as for immunohistochemical characterisation of tumours of neuronal origin. Accepted: 29 July 1997     

Functional analysis of OsTUB8, an anther-specific β-tubulin in rice

Microtubules play important roles in many cellular processes, such as cell division and cell elongation in plants. β-tubulins (TUB), which are one of the basic components of microtubules, are encoded by multigene family in eukaryotes and their nucleotide sequences are highly conserved in protein coding regions. OsTUB8 that was expressed in rice anthers was characterized with a multi-level approach. At the protein level, OsTUB8 was expressed mainly in anthers compared to callus, root, leaf sheath and leaf blade. In situ hybridization and GUS fusion analysis revealed that OsTUB8 was expressed in vascular bundles of anther filaments and in pollen. OsTUB8 expression was lower in the anthers of GA-deficient mutants, ‘Tanginbozu’ and ‘Akibrarewaisei’, compared to those of their respective wild types. Transgenic rice expressing OsTUB8 in an antisense orientation were suppressed in the amount of seed set upon maturity. Antisense-transgenic rice plants were 20–60% shorter compared to the vector-only control. These results suggest that OsTUB8 might be differentially expressed in rice anthers due to the action of GA, and involved in the processes of vegetative growth and seed set in rice.     

Distinct subcellular localization of β-tubulin epitopes in the adult mouse brain

 A panel of monoclonal antibodies specific of α-tubulin (TU-01, TU-09) and β-tubulin (TU-06, TU-13) subunits was used to study the location of N-terminal structural domains of tubulin in adult mouse brain. The specificity of antibodies was confirmed b immunoblotting experiments. Immunohistochemical staining of vibratome sections from cerebral cortex, cerebellum, hippocampus, and corpus callosum showed that antibodies TU-01, TU-09, and TU13 reacted with neuronal and glial cells and their processes, whereas the TU-06 antibody stained only the perikarya. Dendrites and axons were either unstained or their staining was very weak. As the TU-06 epitope is located on the N-terminal structural domain of β-tubulin, the observed staining pattern cannot be interpreted as evidence of a distinct subcellular localization of β-tubulin isotypes or known post-translational modifications. The limited distribution of the epitope could, rather, reflect differences between the conformations of tubulin molecules in microtubules of somata and neurites or, alternatively, a specific masking of the corresponding region on the N-terminal domain of β-tubulin by interacting protein(s) in dendrites and axons. Accepted: 11 November 1996     

Glucose inhibition of galactose-induced synthesis of β-galactosidase in Streptomyces violaceus

Various carbon compounds inhibited galactose induced synthesis of a -galactosidase activity in Streptomyces violaceus. Glucose and 2-deoxyglucose, but not methyl--d-glucose, caused inhibition of galactose uptake activity. In addition, glucose, or one of its metabolites, inhibited the synthesis of the glactose uptake system. Therefore it is concluded that the main inhibitory activity of glucose on galactose induced enzyme synthesis is exerted through inducer exclusion. Other carbon sources, such as d-ribose, d-gluconate, cellobiose or dl--glycerophosphate, did not inhibit uptake of the inducer galactose and may exert their effect through catabolite repression, inactivation or direct enzyme inhibition.     

Differential expression of GAPDH and β-actin in growing collateral arteries

Housekeeping genes like glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and -actin are often used as internal standards for quantitative RNA analysis. In our study we analyzed the relative expression level of GAPDH and -actin as well as of the 18S rRNA and the Poly (A)⁺ RNA in growing collateral arteries in a rabbit model of arteriogenesis which is not associated with ischemia. Relative quantitation of the housekeeping genes displayed a significant upregulation of the -actin- and GAPDH mRNA during the first 24 h of vessel growth. For day 3 our results revealed an even stronger upregulation of the -actin mRNA (140%) but a significant downregulation of the GAPDH mRNA (50% of control). The 18S rRNA, however, showed for the same periods only minor alterations compared to the Poly (A)⁺ RNA. From these results we conclude that the 18S rRNA, but not the GAPDH- or -actin mRNA is an appropriate internal control for relative quantitation of gene expression under conditions of cell proliferation in growing vessels.     

Computational study of binding of epothilone A to β-tubulin

Understanding the interactions of epothilones with β-tubulin is crucial for computer aided rational design of macrocyclic drugs based on epothilones and epothilone derivatives. Despite numerous structure-activity relationship investigations we still lack substantial knowledge about the binding mode of epothilones and their derivatives to β-tubulin. In this work, we reevaluated the electron crystallography structure of epothilone A/β-tubulin complex (PDB entry 1TVK) and proposed an alternative binding mode of epothilone A to β-tubulin that explains more experimental facts.     

The use of 1-O-sulfonyl-d-mannopyranose derivatives in β-d-mannopyranoside synthesis

Several 1-O-sulfonyl derivatives of d-mannopyranose having a nonparticipating benzyl ether group at C-2 and ester functions at C-6 and C-4 were synthesized from the corresponding d-mannopyranosyl chloride derivatives with silver sulfonates in acetonitrile. The reaction of 1-O-sulfonyl-d-mannopyranose compounds with methanol in various solvents at room temperature gave high yields of glycosides with low degrees of stercoselectivity. On the other hand, 1-O-suffonyl-d-mannopyranose derivatives having an acyl participating-group at O-2 and benzyl ethers at C-3, C-4, and C-6 gave high yields and high stereoselectivity of α-d-mannopyranosides with primary and secondary alcohols in several solvents. Model studies were carried out to determine the best combination of 2-O-acyl group, solvent, time, temperature, and 1-O-sufonyl group to give high yields with high stereoselectivity. The method has been used to prepare in good yields more complex glycosides, including perbenzylated methy 2-O-(α-d-mannopyranosyl)-α-d-mannopyranoside.     

Molecular Characterization of β-tubulin gene from Pleurotus sajor-caju

A β-tubulin gene (TUB1) from the basidiomycete Pleurotus sajor-caju was sequenced. TUB1 encodes a 446-amino-acid protein. The coding region is interrupted by 9 introns, all of which had a 5'-GTRNGT…YAG-3' sequence at the boundaries. Locations of the introns in TUB1 were common between the β-tubulin genes of other basidiomycetes, but not with animals, ascomycetes, or plants. This suggests that the introns were inserted independently into the β-tubulin gene after these divisions had diverged.